The histological study of tissues taken by biopsy are of great use in clinical practice, for example for diagnosing tumors. However, this technique is slow and complex to implement, because it requires a biopsy, that is to say the taking of a sample of the tissue to be studied, and for it to be cut into thin slices which are observed by microscope and analyzed by an anatomical pathologist. The full procedure also requires the fixing of the sample, the occlusion thereof in a matrix and the coloring thereof. That poses a problem, above all in the case of examinations made during surgical operations, where speed is of primordial importance. Furthermore, the sampling step can be annoying, even dangerous, for the patient (in the case of the brain for example). For this reason, non-intrusive—notably optical—imaging techniques have been developed to view the internal structure of the biological tissues—or more generally of semi-transparent objects. To be able to compete with the conventional histological examinations, these techniques have to make it possible to access in situ to a depth of the order of a millimeter below the surface of the tissue and exhibit a resolution of the order of a micrometer. The speed of execution, the simplicity and the cost are also important parameters to be taken into account.
None of the imaging techniques known from the prior art gives full satisfaction.
Scanning optical coherence tomography (OCT) is a technique based on “white” (wide band) light interferometry. In its version in the time domain, a beam of white light is divided into two parts, one focused on the tissue to be studied and the other on a reference mirror. The light reflected (backscattered) by the observed object is combined with that reflected by the reference mirror and detected by a photodetector. An interference occurs only when the optical path difference is at most of the order of the coherence length of the radiation; by modifying the optical length of the reference arm of the interferometer, different depths are accessed in the object. An image in 2 or even 3 dimensions can be constructed using interferometry (which allows acquisition according to the axial dimension, that is to say the depth) and scanning (which allows acquisition according to one or two lateral dimensions). In scanning OCT in the frequency domain, the reference arm has a fixed optical length and the interferometric signal is spectrally analyzed. In this respect, see the article by A. F. Fercher “Optical coherence tomography—principles and applications”, Reports on Progress in Physics 66 (2003) 239-303. In practice, only with difficulty does OCT make it possible to obtain lateral resolutions better than approximately a few micrometers.
A more recent technique, full field OCT, uses a two-dimensional image sensor to detect the interferometric signals. This technique, coupled with the use of a light source of low temporal and spatial coherence such as a halogen lamp, makes it possible to substantially improve the spatial resolution—both laterally and depthwise (axially) compared to scanning OCT. However, this technique is ill-suited to applications in which the object is likely to move (particularly for in vivo applications), then leading to scrambling of the interferometric signals. Moreover, it provides “face wise” sections (parallel to the surface of the observed object), whereas vertical sections are often more useful. Furthermore, its depth of penetration is less than in scanning OCT. This technique is described, for example, in the document EP1364181 and in the article by A. Dubois, K. Grieve, G. Moneron, R. Lecaque, L. Vabre, and A. C. Boccara “Ultrahigh-resolution full-field optical coherence tomography”, Applied Optics 43, p. 2874 (2004).
The article by S. Kim et al. “Simultaneous measurement of refractive index and thickness by combining low-coherence interferometry and confocal optics”, Optics Express, Vol. 16, No. 8, 5516 (14 Apr. 2008) describes a method which combines confocal optics and interferometry with low coherence length to characterize a sample by determining its thickness and its refractive index. It is not an imaging method, much less tomographic.
Confocal microscopy uses a spatial filtering to select the light originating from a small region of the observed object; a two- or three-dimensional image can then be reconstructed by scanning. The document EP 2 447 754 describes a slit chromatic confocal microscopy device and method. This system requires:                an illumination in polarized light        an objective having strong chromatic aberrations; and        a spectrometer for measuring the spectrum of the light at the output of the microscope, this measurement making it possible to access the depth probed in the object.        
In this device, the role of the slits is to generate spectral lines (their width defining the spectral resolution and therefore the spatial resolution depthwise in the object). They do not have a confocal filtering role.
The document EP 1 586 931 describes another slit confocal microscopy device and method, which simplifies the process of image reconstruction by allowing for the simultaneous acquisition of a number of pixels along a line. The confocal microscopy, used without fluorescent markers, offers a depth of penetration substantially less than in scanning OCT and in full-field OCT.
The article by Yu Chen et al. “High-resolution line-scanning optical coherence microscopy”, Optics Letters Vol. 32, No. 14, 15 Jul. 2007, pages 1971-1973 describes an apparatus and a method combining slit confocal microscopy and scanning OCT that makes it possible to obtain “face wise” sections of a sample with a higher sensitivity than in full-field OCT. The axial resolution achieved is approximately 3 μm and the lateral resolution approximately 2 μm, these results being obtained by using a very costly femtosecond pulsed laser as light source.
The nonlinear microscopy techniques (two-photon, harmonic generation and other such forms of microscopy) exhibit performance levels—in terms of depth of penetration, spatial resolution and acquisition rate—comparable to those of full-field OCT but at the price of a higher cost and generally longer acquisition times.
The cost and complexity of implementation are also among the main drawbacks of the non-optical imaging techniques, such as X microtomography (which also exhibits a low acquisition rate) and magnetic resonance imaging MRI (the spatial resolution of which is mediocre compared to the optical methods).